High level constitutive production of anthrax protective antigen

ABSTRACT

The present invention relates to a process for preparing anthrax protective antigen protein from  E. coli  using fed batch culture. This process creates a constitutively expressing system for rapid, efficient, cost-effective and high-level production of anthrax PA from  E. coli . The steps of the process involves, transforming  E. coli  DH5α cells with the recombinant constitutive expression plasmid containing the PA gene to obtain recombinant DH5α cells and testing the PA expression by lysis of said cells followed by denaturing gel electrophoresis and Western Blotting technique using PA antibodies. This is followed by fermentation and harvesting of the high cell density cells. The said cells are solubilized using 6-8 Molar Urea and separated by centrifugation. The urea denatured PA is isolated from said supernatant and purified and thereafter eluted.

FIELD OF THE INVENTION

This invention relates to high level constitutive production of anthraxprotective antigen in E. coli by fed batch culture.

BACKGROUND OF THE INVENTION

Anthrax a zoonotic disease is caused by Gram positive, sporulatingbacteria, Bacillus anthracis. Protective antigen, PA is the majorcomponent of all the vaccines against anthrax. Till date, culturesupernatants of B. anthracis have been the major source of purifying PA.However, working with B. anthracis cultures requires P3 facilities thatare cost-prohibitive. Apart from this, PA preparation from B. anthracisis often contaminated with other anthrax toxin proteins. Researchershave tried expressing and purifying PA in other microorganisms such asBacillus subtilis, Baculovirus and E. coli. Purification of PA fromBacillus subtilis resulted in poor yields, required growth in richmedia, and enormous amount of PA was degraded due to proteases secretedby the organism (L. W. J. Baillie et al, Lett. Appl. Microbial (1994)19, 225-227). Baculovirus vectors expressed PA in insect cells; however,purification could not be possible due to low yields. Although PA hasbeen expressed in E. coli, attempts to overproduce the protein were notsuccessful (M. H. Vodkin et al, Cell, (1983) 34, 693-697). Researchersalso purified PA by guiding the protein to the periplasmic spaces,however the yields of the purified PA were very low. All the knownexpression systems for Protective Antigen expression using E. coli, areinducible systems that require the use of IPTG, an expensive chemical.

U.S. Pat. No. 2,017,606 describes the preparation of anthrax antigen bygrowing Bacillus anthracis on a suitable culture medium and separatingthe bacilli from the culture medium.

U.S. Pat. No. 2,151,364 describes a method of producing an anthraxvaccine which comprises preparing the suspension of anthrax spores andadding to the suspension a sterile solution containing alum.

The drawbacks in the above US patents is that both of them use Bacillusanthracis cultures or spores. Bacillus anthracis is an infectiousorganism and cannot be handled without containment facilities. Thelevels of protective antigen expressed in Bacillus anthracis are verylow. This kind of vaccine preparation is also contaminated with othertoxic and non-toxic proteins from Bacillus anthracis resulting in anumber of side-effects and reactogenicity.

The object of this invention is to create a constitutively expressingsystem for rapid, efficient, cost-effective and high-level production ofanthrax PA from E. coli using Fed-Batch culture.

To achieve the said objective this invention provides a process forpreparing anthrax protective antigen protein from E. coli using fedbatch culture comprising:

-   -   transforming E. coli DH5α cells with the recombinant        constitutive expression plasmid containing the PA gene to        produce the recombinant DH5α cells expressing the PA protein,    -   growing said recombinant DH5α cells and testing the PA        expression by lysis of said cells followed by denaturing gel        electrophoresis and Western Blotting technique using PA        antibodies,    -   fermenting said cells in a bio-reactor using:        -   polyols, carbohydrates or organic acids as primary            supplements in Luria Broth medium at 32-42° C.,        -   fed-batch culture technique, and        -   pH-DO stat method of sensing nutrient deprivation to produce            high cell density culture expressing PA protein,    -   harvesting said cells by centrifugation of said high cell        density culture at 5000-10,000 rpm for 10-30 minutes,    -   solubilizing said high cell density culture cells by using 6-8        Molar Urea solution and stirring at ambient temperature for 1-2        hours,    -   separating said high cell density culture debris by        centrifugation at 10,000-15,000 rpm for 30-60 minutes at        32-42° C. and collecting the supernatant containing urea        denatured PA,    -   isolating said urea denatured PA from said supernatant and        purifying it by Ni-NTA chromatography by gradual removal of urea        while said PA is bound to the affinity column, and    -   eluting said purified renatured PA and storing protective        antigen (PA) protein as frozen aliquots at −20 to −70° C.        depending upon immediate or long term use.

The said recombinant constitutive expression plasmid used expresses thePA protein as insoluble inclusion bodies in the E. coli strain DH5αcells.

The harvesting of said cells by centrifugation of said high cell densityculture is carried out at 5000 rpm for 10 minutes.

The centrifugation of said high cell density culture debris is carriedout at 10,000 rpm for 30 minutes for maximizing the harvesting of saidcells.

The said polyol used as primary supplement in Luria Broth medium duringfermentation is glycerol,

The said carbohydrates used as primary supplement in Luria Broth mediumare glucose, galactose, maltose, fructose and lactose,

The said organic acid used as primary supplement in Luria Broth mediumis malic acid.

By using polyol, carbohydrate or organic acid, as primary supplement inLuria Broth medium, the maximum cell density ranges between 10-14optical density units in shake flask cultures.

The maximum cell density of the recombinant cells is achieved by FedBatch cultures containing MgSO₄.

The concentration of Luria Broth medium used in the feed is 5-25×.

The concentration of Luria Broth medium used in the feed is 25× in orderto minimize the volume of feed added during fermentation.

The said plasmid is pQE series vector containing an E. coli recognizablephage promotor.

An anthrax antigen comprises of purified structurally, biologically andfunctionally active recombinant protective antigen (PA) protein ofBacillus anthracis expressed as a 6× histidine fusion protein in E. coliDH5α cells free from polysaccharides, dead bacteria, culture medium,water-soluble and insoluble by-products and suspended impurities.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described with reference to the followingprotocol and the accompanying drawings.

FIG. 1 shows that the maximum optical density in shake flask culture isobtained by using glucose as the additional carbon source and theminimum optical density is obtained by using maltose.

FIG. 2 shows that the optical density obtained in batch and Fed Batchcultures with and without the use of MgSO4.

FIG. 3 shows that the recombinant PA produced is biologically andfunctionally as active as native PA from Bacillus anthracis.

DETAILED DESCRIPTION

The recombinant constitutive expression plasmid containing the PA genecloned in a pQE series vector was used for transforming E. coli strainDH5α competent cells. The said cells containing the recombinant plasmidwere grown overnight at 37° C. and 250 rpm in Luria broth with 100 μg/mlof ampicillin. Cells were harvested by centrifugation at 5,000 rpm for20 minutes. Expression and localization of PA was confirmed by SDS-PAGE.More than 90% of the recombinant protein was found to be present ininclusion bodies. The amount of protein expressed was found to increasein direct proportion to the increase in cell density of the growingculture.

Complex media of modified 100 ml LB of 5× concentrations each wasprepared with glucose, fructose, galactose, lactose, maltose, malicacid, and glycerol as seven different carbon sources in shake flasks.Only LB without any additional carbon source was used as a control.Equimolar amounts of carbon atoms were used as a carbon source and theconcentration was kept equivalent to 0.5% glucose. 10 mM MgSO₄, 100 mMpotassium phosphate and trace elements were also added. All the flaskswere inoculated at the same time with an overnight grown inoculum ofDH5α cells containing the said recombinant plasmid. Samples werecollected aseptically-every hour and OD₆₀₀ was measured. Culturealiquots were collected and their recombinant protein content wasanalyzed by SDS-PAGE. The highest OD₆₀₀ (Optical Density at 600nanometers) could be achieved where glucose was used as the main carbonsource and the lowest OD₆₀₀ was seen in the case of LB and maltose (FIG.1). Maximum protein concentration was obtained by the use of malic acid,maltose and glycerol as the main carbon source. The lowest proteinconcentration was seen in the case of LB and lactose.

A 5 L Biostat B (B Braun Biotech International) fermentor equipped withpH, temperature, dissolved oxygen, and antifoam probes was used for thefermentation runs. The fermentor was interfaced with a personalcomputer. MFCS/win 2.0 software was used for data acquisition andoperation of the fermentor in both batch as well as fed-batch mode.

The medium used for fermentation was a complex media consisting of LuriaBroth with MgSO₄.7H₂O (10 mM), potassium phosphate (5 g/l) and glycerol(1%) at pH 7.4. For fed batch cultures, MgSO₄ was added to the mediumbefore autoclaving. Glycerol was also added to the medium beforeautoclaving. A 25× feed was prepared with 25% glycerol (w/v) and 25× LBand autoclaved separately. Potassium phosphate solution was alsoautoclaved separately, allowed to reach the room temperature and addedaseptically just before starting the run. The pH probe was calibratedwith standard buffers of pH 7.0 and 9.2 before autoclaving. Afterautoclaving the fermentor media was automatically adjusted to pH7.4 bythe addition of 1N NaOH/1 MHCl and temperature was set to 37° C. The DO(Dissolved oxygen) probe was calibrated by setting the electronic zerovalue of the dissolved oxygen in the range of zero to +15 nano-amperes(nAmp) and 100% DO value was given to oxygen tension of 2 vvm of pumpedair, at an agitation rate of 250 rpm. The fermentor was started in batchphase with a working volume of 2 L. The recombinant plasmid containingcells were grown overnight on LB under the selective pressure of 100μg/ml of ampicillin, at 37° C., 250 rpm. 1% of the overnight grownculture was used to inoculate the fermentor. The DO value was set at 40%and the stirrer was shifted to the cascade mode. In this mode, followingthe inoculation, as the DO begins to fall below 40%, the stirrer speedincreases automatically to maintain the value at 40%. Samples werecollected at an interval of 1 hour. The culture aliquots were diluted toan optical density (OD₆₀₀) approximately below 0.5 units. As the growingculture reached the mid log phase, feeding was started from the 25× feedusing a peristaltic pump (Pharmacia). The feed rate was monitored andmanually controlled to maintain a pH value between 7.2 and 7.4. Oxygensupplementation became necessary after an OD of 70 was reached. Oxygenwas supplied to the culture by using the Gasmix function of thefermentor. The culture was grown to an OD₆₀₀ of 120 units after whichthe cells were harvested. Antifoam was initially added to the mediumbefore autoclaving and later it was added as and when required.

To optimize the media composition and other conditions for thesuccessive fermentation runs, a series of batch runs were carried out onmodified LB with glycerol with or without the incorporation MgSO₄ in thegrowth medium. The batch runs without MgSO₄ in modified LB (withglycerol as carbon source) could attain the maximum OD of ˜14 whereasthe batch runs carried out with MgSO₄ in modified LB could attain OD₆₀₀values of more than 18. It was inferred that MgSO₄ is necessary for thegrowth of cells at higher densities. The incorporation of MgSO₄ in thegrowth medium is essential for achieving higher biomass yields duringfermentation. (FIG. 2).

5 ml of the high cell density culture was centrifuged at 10,000 g forhalf an hour in pre weighed tubes. After draining the supernatant, thetube with the centrifuged cells was weighed on a balance to determinethe wet cell weight. To determine the dry cell weight, the same tube wasleft overnight in an incubator at 70° C. and was weighed next day.

To check the stability of the plasmid pMW1 in the fermentor, culturesamples were aseptically collected every hour during the fermentationrun. The samples were diluted such that the OD₆₀₀ of each sample was˜5.0 and centrifuged at 10,000 g for 1 minute. The supernatant wasdrained completely and the pellet was suspended in 100 μl lysis buffercontaining 100 mM potassium phosphate buffer, pH8.0 with 8M urea. Thesesamples were subjected to SDS-PAGE to check for the protein expressionfrom the recombinant plasmid. Colony preparations and minipreparationsof plasmid DNA were also made to directly check the presence of therecombinant plasmid inside the expressing cells. No noticeablegeneration of plasmid free cells was seen.

The protein was purified using metal-chelate affinity chromatographyunder denaturing conditions. In brief the pellet from 100 ml culture wasresuspended in 100 ml of denaturing buffer containing 100 mM sodiumphosphate buffer, 300 mM sodium chloride and 8M urea (pH 8.0). Theresuspended pellet was incubated at 37° C. for 2 hrs. on a rotaryshaker. The lysate was centrifuged twice for 60 min. each at roomtemperature and the supernatant was mixed with 50% Ni-NTA slurry. Theslurry was packed into a column and allowed to settle. The, flow throughwas reloaded on the column. Ni-NTA matrix was washed with 500 mldenaturing buffer containing 8M urea, followed by on-column renaturationof the protein using 8M-0M Urea gradient. The protein was eluted with250 mM Imidazole chloride in elution buffer, 100 mM sodium phosphate ofpH 8.0 with 250 mM imidazole and 300 mM sodium chloride. 10 μl of eachfraction was analyzed on 12% SDS-PAGE. Fractions containing the proteinwere collected, pooled and dialyzed against 10 mM HEPES buffercontaining 50 mM NaCl and stored frozen at −70° C. in aliquots.

The specific protein was estimated by a number of methods. Densitometrywas done using BioRad gel documentation system and the Quantity Onesoftware. The fold purification of PA was determined at different stagesby calculating the amount of protein required to kill 50% of J774A.1macrophage-like cells (EC₅₀) in combination with LF (1 μg/ml) for 3 hrsat 37° C. The purified protein was measured by Bradford's method andalso by determining the OD of the preparation at 280 nm. PA accountedfor more than 30% of the total cell protein and was present in the cellsin the form of inclusion bodies. 5-8 g/L of PA was produced inside thecells.

The biological activity of the rPA (recombinant PA) was also determinedby the cytotoxicity assay on J774A.1 macrophage like cell line. Allexperiments were done in triplicates. In brief varying concentrations ofrPA protein alongwith LF (1 μg/ml) was added to the cells. The native PAfrom B. anthracis alongwith LF was kept as the positive control. After 3hrs., cell viability was determined using MTT dye and the resultingprecipitate was dissolved in a buffer containing 0.5% (w/v) sodiumdodecyl sulfate, 25 mM HCl in 90% isopropyl alcohol. Absorption was readat 540 nm using a microplate reader (BioRad) and percent viability wasdetermined. It was found that rPA alongwith LF was fully able to lysemacrophage cells and its biological activity was similar to PA preparedfrom B. anthracis. The EC₅₀ of rPA and nPA was found to be ˜50 ng/mleach (FIG. 3) The fold purification of the protein was determined ateach stage of purification by the above-mentioned cytotoxicity assays.(Table 1)

TABLE 1 Purification of PA from Escherichia coli Volume ActivityPurification Fraction (ml) Protein (mg/ml) (EC₅₀)^(a) (fold)^(b) Celllysate 10 291 57.3 1 Affinity 25 1.8 0.0241 1965 Purification ^(a)EC₅₀is defined as the concentration of PA (μg/ml) along with LF (1 μg/ml)required to kill 50% of the J774A.1 cells. After 3 hrs. of incubation,viability was determined by MTT dye. The results represent the mean ofthree experiments. ^(b)Purification fold was determined by dividing EC₅₀for cell lysate with EC₅₀ for fractions obtained from different columns

DISCUSSION

The overexpression of any recombinant protein depends upon the optimumconfiguration of the various elements of the expression system. Severalfactors drastically affect recombinant protein expression like-promoterstrength, plasmid stability, plasmid copy number, transcriptionterminators, transcription and translation efficiency that ultimatelyenhances mRNA stability, translation terminators, tight regulation ofgene transcription, availability of ribosomes, post translationalmodifications, the stability and solubility of the recombinant proteinitself, as well as host cell and culture conditions. Processes aiming athigh levels of heterologous protein expression make use of strongpromoters like T5, T7, PL, PR, Ptrc, Ptac, to create an effectiveexpression system. Most of such systems carry inducible promoters andinduction is preceded by a phase of low or no product formation. Uponinduction, the specific production rate increases to a maximum within ashort time period and the protein is continuously produced for 4-5hours. Following induction, a number of changes have been reported likea change in carbon metabolism that results in acetate accumulation, anincrease in respiratory activity, decrease in the synthesis of housekeeping proteins, and occurrence of a heat shock-like response and SOSresponse. All of these responses contribute towards degradation ofrecombinant products. The formation of inclusion bodies prevents therecombinant protein from the onslaught of cellular proteases, whichotherwise may result in extensive degradation of the protein leading tolow product recoveries. The SDS-PAGE and Western blot analysis atdifferent time points, corroborates the fact that expression of PA fromthe recombinant plasmid pMW1 is not leaky and is proportional to the ODof the culture. With no significant degradation, most of the protein waspresent inside the cells in the form of inclusion bodies.

Once a strong expression system is created, the recombinant proteinproduction in E. coli can be increased significantly by the use of highcell density culture employing different techniques. Cell concentrationsof more than 50 g/l of dry weight can be routinely achieved to providecost effective production of recombinant proteins. But high cell densitycultures also have a few drawbacks such as substrate inhibition, limitedoxygen transfer capacity, the formation of growth inhibitoryby-products, and limited heat dissipation resulting in reduction in themixing efficiency of the fermentor. A major challenge in the productionof recombinant proteins at high cell density culture (HCDC) is theaccumulation of acetate, a lipophilic agent that is detrimental to cellgrowth. The accumulation of acetate in the growth medium is reported toreduce recombinant protein production. A number of strategies have beendeveloped to reduce acetate formation in fed batch culture likecontrolling the specific growth rate by limiting essential nutrients ofmedia such as carbon or nitrogen source, varying growth conditions andE. coli strains. Since the primary goal of fermentation research iscost-effective production of recombinant products, it is important todevelop a cultivation method that allows the maximization of the yieldsof the desired product. The composition of the growth media is crucialfor enhancing product formation as well as acetate reduction. Acetate isproduced in E. coli under oxygen limiting conditions or in presence ofexcessive glucose under aerobic conditions, when carbon flux in thecentral metabolic pathway exceeds its biosynthetic demand and thecapacity for energy generation within the cell.

On the basis of the growth kinetics and recombinant protein yields,glycerol was selected as the main carbon source for HCDC out of 7 othercarbon sources namely, glucose, fructose, lactose, galactose, maltose,malic acid, and glycerol. Acetate is not produced when glycerol is usedas a carbon source and high cell densities may be achieved relativelyeasily using glycerol. The lower rate of glycerol transport into thecell, compared with that of glucose, apparently leads to a reduction inthe flux of carbon through glycolysis; greatly reducing acetateformation and the cells grow more slowly on glycerol. Glycerol also hasan anti foaming effect that leads to less frothing during the course ofthe fermentation run.

The method utilized for nutrient feeding is also crucial for the successof HCDC as it affects both the cell density and cell productivity.Constant or intermittent feeding is carried out under nutrient limitingconditions. Although other feeding strategies have been successfullyemployed to HCDCs in E. coli, more sophisticated feeding strategies withfeed-back control schemes have been developed lately. The feeding rateis coupled with other physical parameters such as DO (dissolved oxygen),pH microbial heat and CO₂ evolution rate (CER). The DO-stat method offeeding is based on the fact that the DO in the culture increasessharply when the substrate is depleted. The cells are not able to growrapidly as the nutrient levels go down and lesser oxygen is utilized bythe cells. Therefore, in the DC-stat method, the substrate concentrationis maintained within the desired range by automatically adding anutrient when the DO rises above the preset value. Another option, thepH-stat method is based on the fact that the media pH changes when theprincipal carbon source becomes limiting. When the carbon source isexhausted, the pH begins to rise mainly as a result of increase in theconcentrations of ammonium ions excreted/secreted by the cells.

Analysis of the feeding system starts with characterization of theprobing and detection method. The idea is to detect the saturation inthe respiration by checking the DO and pH responses to pulses in thefeed rate. Once feeding is initiated and E. coli enters into log phase,the feed is consumed more or less in an exponential manner. But, thefeeding rate has to be controlled so that it doesn't exceed the nutrientdemand or feed consumption rate. It is done by maintaining the pH and DOaround their set values. A fall in pH and DO is an indication ofsubstrate overdosing. Rise in pH and DO values indicate that the carbonsource or one of the substrates is limiting and hence feed is required.If the increase in feed pulse/rate is unable to generate any significantresponse, i.e. fall in DO or increase in the stirrer speed, it is aperfect indication that some other factor like MgSO₄, KH₂PO₄ or traceelements has become limiting and has to be added intermittently on suchoccasions. The addition is followed by a rapid change in theabove-mentioned variables. E. coli is able to utilize acetate as acarbon source when glucose or any other principal carbon source isabsent. The consumption of acetate is characterized by a deviation fromthe preset values to lower pH values and cyclic patterns start appearingin the consumption of oxygen, till the preset pH value is graduallyregained by the culture. At this time feeding is restarted.

The DO-stat method responds more rapidly to nutrient depletion than thepH-stat method. When complex substrates are used together withcarbohydrate substrates, the DO change is not as apparent as the cellscontinue to use the complex substrates. The feeding strategy used in thepresent experiments was a combination of both the pH-stat as well asDO-stat methods. Monitoring of both the parameters simultaneously givesbetter control over the growth conditions of the growing culture. Makinguse of this strategy of substrate feeding we were able to achieve an ODof 120 which is a greater than six-fold increase from the OD achieved inthe batch runs and more than 23-fold from that in the shake flaskcultures.

This is the first report on optimization of fed-batch HCDC conditions toachieve high yields of PA in E. coli. This work is an attempt to obtaina large amount of non-reactogenic PA that could serve as a prospectivevaccine candidate. The method of PA production reported here utilizes anovel and advantageous substrate and a simple and easy to controlfeeding strategy that may also be successfully applied to otherrecombinant protein expression systems to achieve high product yields.

The invention claimed is:
 1. A process for preparing anthrax protectiveantigen protein from E. coli. using fed batch culture comprising thesteps of (a) transforming E. coli DH5α cells with a recombinantconstitutive expression plasmid containing the Protective Antigen geneto produce recombinant DH5α cells expressing the Protective Antigenprotein, (b) growing said recombinant DH5α cells and testing ProtectiveAntigen expression by lysis of said recombinant cells followed bydenaturing gel electrophoresis and a Western Blotting technique usingProtective Antigen antibodies, (c) fermenting said recombinant cells ina bio-reactor in Luria Broth medium at 32-42° C. in a fed batch culture,wherein the medium comprises nutrients, including a primary supplementselected from any one or all of a polyol, a carbohydrate and an organicacid, and wherein the fermenting comprises simultaneously monitoring adissolved oxygen concentration and pH of the medium and, as nutrientsare depleted from the medium, adding replacement nutrients to the mediumto maintain the dissolved oxygen concentration and pH at levels thatresult in attainment of a high cell density culture that expressesProtective Antigen in a yield of at least 5 g/l, (d) harvesting saidfermented cells by centrifugation of said high cell density culture at5000-10,000 rpm for 10-30 minutes, (e) solubilizing said high celldensity culture cells by using 6-8 Molar Urea solution and stirring atambient temperature for 1-2 hours, (f) separating high cell densityculture debris by centrifugation at 10,000-15,000 rpm for 30-60 minutesat 32-42° C. and collecting supernatant containing urea denaturedProtective Antigen, (g) isolating said urea denatured Protective Antigenfrom said supernatant and purifying it by NI-NTA chromatography bygradual removal of urea with said Protective Antigen bound to anaffinity column whereby to form purified renatured Protective Antigen,and (h) eluting the purified renatured Protective Antigen and,optionally, storing Protective Antigen protein as frozen aliquots at −20to −70° C.
 2. A process as claimed in claim 1, wherein said recombinantconstitutive expression plasmid expresses the Protective Antigen proteinas insoluble inclusion bodies in the E. coli strain DH5α cells.
 3. Aprocess as claimed in claim 1, wherein harvesting of said cells bycentrifugation of said high cell density culture is carried out at 5000rpm for 10 minutes.
 4. A process as claimed in claim 1, whereincentrifugation of said high cell density culture debris is carried outat 10,000 rpm for 30 minutes for maximizing the harvesting of saidcells.
 5. A process as claimed in claim 1, wherein said polyol usedduring fermentation is glycerol, as primary supplement in Luria Brothmedium at 37° C.
 6. A process as claimed in claim 1, wherein saidcarbohydrate is selected from the group consisting of glucose,galactose, maltose, fructose, and lactose, as primary supplement inLuria Broth medium at 37° C.
 7. A process as claimed in claim 1, whereinsaid organic acid is malic acid, as primary supplement in Luria Brothmedium at 37° C.
 8. A process as claimed in claim 1, wherein by usingpolyol, carbohydrate or organic acid, as primary supplement in LuriaBroth medium, the maximum cell density ranges between 10-14 opticaldensity units in shake flask cultures.
 9. A process as claimed in claim1, wherein the maximum cell density of the recombinant cells is achievedby a Fed Batch culture containing MgSO₄.
 10. A process as claimed inclaim 1, wherein the Luria Broth medium is fed into the bioreactor at5-25 times dilution concentration of from an initial concentration ofthe Luria Broth medium.
 11. A process as claimed in claim 1, wherein theLuria Broth medium is fed into the bioreactor at 25 times dilutionconcentration of 25X from an initial concentration of the Luria Brothmedium.
 12. A process as claimed in claim 1, wherein said plasmid is pQEseries vector containing an E. coli recognizable phage promotor.
 13. Theprocess as claimed in claim 1, wherein the process consists essentiallyof said steps (a)-(h).